A DNA Barcode Library for North American Ephemeroptera: Progress and Prospects

Jeffrey M. Webb1*, Luke M. Jacobus2, David H. Funk3, Xin Zhou4, Boris Kondratieff5, Christy J. Geraci6,R. Edward DeWalt7, Donald J. Baird8, Barton Richard9, Iain Phillips10, Paul D. N. Hebert1 1 Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada, 2 Division of Science, Indiana University Purdue University Columbus, Columbus, Indiana, United States of America, 3 Stroud Water Research Center, Avondale, Pennsylvania, United States of America, 4 BGI, Shenzhen, Guangdong Province, China, 5 Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, Colorado, United States of America, 6 Department of Entomology, National Museum of Natural History, Smithsonian Institution, Washington, D. C., United States of America, 7 Prairie Research Institute, Illinois Natural History Survey, University of Illinois, Champaign, Illinois, United States of America, 8 Environment Canada, Canadian Rivers Institute, Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada, 9 Laboratory of Aquatic Entomology, Florida A&M University, Tallahassee, Florida, United States of America, 10 Saskatchewan Watershed Authority, Saskatoon, Saskatchewan, Canada

Abstract DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for water quality assessment programs. A prerequisite for identification using barcodes is a reliable reference library. We gathered 4165 sequences from the barcode region of the mitochondrial cytochrome c oxidase subunit I gene representing 264 nominal and 90 provisional of (Insecta: Ephemeroptera) from Canada, Mexico, and the United States. No species shared barcode sequences and all can be identified with barcodes with the possible exception of some . Minimum interspecific distances ranged from 0.3–24.7% (mean: 12.5%), while the average intraspecific divergence was 1.97%. The latter value was inflated by the presence of very high divergences in some taxa. In fact, nearly 20% of the species included two or three haplotype clusters showing greater than 5.0% sequence divergence and some values are as high as 26.7%. Many of the species with high divergences are polyphyletic and likely represent species complexes. Indeed, many of these polyphyletic species have numerous synonyms and individuals in some barcode clusters show morphological attributes characteristic of the synonymized species. In light of our findings, it is imperative that type or topotype specimens be sequenced to correctly associate barcode clusters with morphological species concepts and to determine the status of currently synonymized species.

Citation: Webb JM, Jacobus LM, Funk DH, Zhou X, Kondratieff B, et al. (2012) A DNA Barcode Library for North American Ephemeroptera: Progress and Prospects. PLoS ONE 7(5): e38063. doi:10.1371/journal.pone.0038063 Editor: Brock Fenton, University of Western Ontario, Canada Received February 2, 2012; Accepted April 30, 2012; Published May 30, 2012 Copyright: ß 2012 Webb et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (2008-OGI-ICI-03), and Environment Canada’s Competitiveness and Environmental Sustainability Indicators (CESI) Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction addition to allowing the identification of difficult specimens, barcoding provides a level of data standardization that has been DNA barcoding [1] of , the analysis of a standardized previously lacking in environmental assessments [13], aiding segment of the mitochondrial cytochrome c oxidase subunit 1 broader comparisons of results gathered through monitoring (COI) gene, has rapidly become an important tool for the programs. Furthermore, barcoding reliably produces species-level identification, delimitation, and discovery of species [1–3]. DNA (or even population-level) identifications that can improve the barcoding has the further advantage that identification success sensitivity of analyses, depending on the aims of the assessment extends across all life stages, allowing the association of immatures program [14,15]. with adults [4,5]. Its capacity to identify all life stages is particularly An essential requirement of biomonitoring, conservation bi- important for aquatic ecology and biological monitoring (biomo- ology, and ecology is a sound of the study organisms. nitoring) of water quality because the aquatic larvae are usually the Although taxa could be based purely on operational taxonomic life stage studied [6]. Unfortunately, this is the life stage that is the units (OTUs) defined by sequence data, information on key most poorly known taxonomically because most species concepts functional niche traits (e.g. [16]; functional feeding groups, life in aquatic are based on the morphology of adult males. The history data, behavior and historical distribution) and environ- identification of larvae is further hindered by the fact that many mental sensitivity [17] requires linking molecular OTUs with are rather delicate, especially mayflies (Ephemeroptera), and the known taxa. structures critical for confident identifications such as gills, legs, Mayflies are abundant in most aquatic habits, and show varied and caudal filaments are commonly damaged or missing. tolerance to differing disturbance regimes, making them ideal The application of DNA barcoding to freshwater biomonitoring candidates for monitoring water quality. Together with the has recently generated much interest for several reasons [7–12]. In caddisflies (Trichoptera) and stoneflies (Plecoptera), they comprise

PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e38063 DNA Barcoding North American Ephemeroptera one of the most commonly used biomonitoring metrics, EPT intraspecific divergences greater than 2.2%, a level of divergence richness [18], and as such are one of the focal groups for the found to delimit species across diverse groups of insects [1,2,20] International Barcode of Life Freshwater Biosurveillance Working although higher maximum intraspecific distances have been Group (www.ibol.org). observed in Trichoptera when widely separated geographic areas The development of a barcode reference library for North are included [3]. Almost 20% of species with more than one American mayflies was initiated by sequencing 150 specimens specimen had maximum intraspecific divergences .5.0%; these from 80 North American species [2]. Those results indicated that species with high intraspecific divergences may represent species- all species tested could be identified accurately using barcodes, but complexes and when they are excluded from the analysis, the revealed several cases of deep sequence divergence within a species. mean maximum intraspecific divergence decreased to 1.3%. In 44 Further species were added [19] and barcoding techniques used as species (12.5%), the maximum intraspecific distance was greater a taxonomic tool to confirm the validity of a presumed new species than the minimum interspecific distance. A Neighbor Joining tree and to support the synonymy of two species of Heptagenia of all specimens is available in Figure S1. (). Additional barcodes for North American mayflies Increasing the geographic range between samples did not were generated from a regional inventory of northeastern always lead to large increases in intraspecific divergence. For Manitoba [20,21], from a test of the efficacy of barcoding for example, vitreus (Walker) was sampled from throughout biological monitoring using aquatic macroinvertebrates [12], and its latitudinal range but the maximum intraspecific divergence a general barcoding paper [1]. Additional barcode sequences for was only 2.8% and a specimen of Ephemera simulans Walker from North American species of Ephemerella have been generated [22], Colorado differed by only a single nucleotide (0.15%) from but because these sequences are not publicly available, we do not a specimen from Churchill, Manitoba, a distance of over consider them further. 2,200 km. Even in species with multiple barcode clusters, The fauna of North America includes 651species geographically distant specimens often clustered within the (Mayfly Central, http://www.entm.purdue.edu/mayfly/na- same group and specimens with small geographic distances species-list.php accessed 30 November 2011), but 10 of these taxa sometimes occurred in different barcode clusters. The cluster of are nomina dubia and four are recently extinct. Ignoring the latter Acentrella parvula (McDunnough) that included a topotypical two groups, 637 species and 8 subspecies remain. Previous papers (from type locality) specimen from southern Ontario, for have assembled barcodes for 121 of these species, most from example, also included specimens from Saskatchewan and eastern North America [1,2,12,19,20]. In this paper, we broaden New Brunswick, but other specimens of A. parvula from New geographic coverage in North America (including Mexico) and Brunswick formed a separate cluster together with specimens raise barcode coverage to more than 350 taxa. Aside from from New York. reporting this progress, we provide guidance for further barcoding efforts on this group and highlight taxonomic problems in the Barcode Divergences Between Species North American Ephemeroptera. Among the morphologically distinct species Siphlonurus rapidus McDunnough, S. typicus (Eaton), and S. sp.JMW1 the minimum Results divergence between species was 1.3–1.6%, but each species was Although most specimens derived from east of the Rocky monophyletic and so can be distinguished by barcode sequences Mountains, collections were made across North America using a tree-based criterion. Furthermore, each of the species can (Figure 1). Sequences (average length = 646 bp, ranging from be identified by 2–6 fixed nucleotide differences (Table 2). Among 314–658 bp) were obtained from 4165 specimens; 3024 are newly Caenis amica Hagen, C. punctata McDunnough, and C. youngi analyzed and 1141 derive from prior studies. These sequences Roemhild, the interspecific distances were as low as 0.3% provide coverage for 71 of 106 genera and 18 of 21 families known (maximum intraspecific distances ranged from 3.7–21.9% and from North America. In addition, these records provide coverage none of the species were monophyletic), possibly an artifact of for 264 of the 647 species known from North America and for incomplete taxonomic knowledge or historical introgression. For another 90 provisional species (Table 1). Because many of these nearly all other species, the minimum interspecific distances were provisional species were only represented by subimagos, females, much greater (mean: 12.5%). or larvae that cannot be identified to species using morphological All but 9 of the 44 species with a maximum intraspecific characters, many probably represent named species for which we divergence greater than the minimum interspecific divergence have no sequences from adult males. As a consequence, as much were polyphyletic or paraphyletic. Centroptilum triangulifer as 55% of the North American fauna may have coverage, and (McDunnough), C. minor (McDunnough), Ephoron album (Say), perhaps as much as 60% of the 583 species known from Canada Maccaffertium mexicanum integrum (McDunnough), Siphlonurus rapi- and the United States. Most of these taxa (284/354) were dus, Stenonema femoratum (Say), Eurylophella funeralis (McDunnough), represented by more than one specimen (mean: 11.8, maximum: Ephemerella dorothea infrequens McDunnough, and Teloganopsis 236) (Table S1). deficiens (Morgan) were all monophyletic with a maximum No species shared haplotypes but previously published intraspecific divergence greater than the minimum interspecific sequences for nine currently valid species clustered with specimens divergence. Some of these species are clearly species complexes, assigned to a different taxon. Whenever we were able to with well-defined and deeply divergent clusters with associated reexamine specimens, the original morphological identification morphological differences (i.e. Maccaffertium mexicanum integrum). proved incorrect. Table S2 summarizes these identification The non-monophyletic species are also likely species complexes updates which have now been implemented on GenBank and as preliminary examination of some, such as Baetis tricaudatus BOLD (Barcode of Life Data Systems www.boldsystems.org). Dodds, show morphological variation corresponding to barcode The average maximum intraspecific divergence was 3.9% clusters. Most of the species with very large intraspecific (max = 26.7%) and the average intraspecific divergence was distances have at least one synonym. Further discussion of 1.97%. Minimum interspecific distances ranged from 0.3–24.7% species with high intraspecific divergence and/or polyphyletic (mean: 12.5%). One hundred five species (29.7%) had maximum haplotype groups is available in Text S1.

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Figure 1. Distribution map for all barcoded specimens of Ephemeroptera with geocoordinates (n = 3902). doi:10.1371/journal.pone.0038063.g001

Table 1. Summary of current barcode coverage and divergence values for the 21 families of Ephemeroptera known from North America (NA).

# known NA # barcoded species and # barcoded provisional # species with Family subspecies species species MXID $2.2% Mean MXID Mean MNID

Acanthametropodidae 2 0 0 0 – – Ameletidae 35 20 1 4 1.2 13.6 Ametropodidae 2 1 0 0 0.2 – Baetidae 146 58 37 32 6 15.3 Baetiscidae 11 4 0 2 2.8 9.3 Behningiidae 1 1 0 1 7.9 – 36 12 1 6 8 11.6 72 48 13 21 4.7 11.7 Ephemeridae 13 9 0 3 3.3 8.2 Euthyplociidae 1 0 0 – – – Heptageniidae 130 56 21 19 2.8 10.2 Isonychiidae 15 7 4 2 1.5 13.3 Leptohyphidae 35 6 8 2 2.4 14.1 Leptophlebiidae 88 24 10 9 3.4 14.1 Metretopodidae 9 2 0 1 5.1 14.3 Neoephemeridae 4 1 0 0 0.8 – Oligoneuriidae 8 0 0 – – – Palingeniidae 1 1 0 0 1.6 – Polymitarcyidae 7 2 0 1 4.8 6.3 Potamanthidae 5 1 0 0 0.2 – Siphlonuridae 24 11 5 1 1.8 9.4

All distances are % K2P; MNID = minimum interspecific K2P distance, MXID = maximum intraspecific K2P distance. doi:10.1371/journal.pone.0038063.t001

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Table 2. Diagnostic nucleotides in Siphlonurus rapidus, resolution of this uncertainty lies in the acquisition of DNA S. typicus, and S. sp.JMW1. barcodes from the holotype of each taxon as it represents the only unambiguously identified specimen. Because type specimens of North American Ephemeroptera have a mean age of 75 years Species Diagnostic Nucleotides (position:nucleotide) (range 0–250 years), DNA degradation will be significant. However, prior work on Lepidoptera has shown that DNA Siphlonurus rapidus 364:T, 502:C barcode sequences can regularly be recovered from specimens that Siphlonurus typicus 208:A, 232:C, 574:A are less than a century old by assembling short amplicons [27,28]. Siphlonurus sp.JMW1 79:A, 172:C, 397:T, 433:A, 541:A, 553:A When sequences cannot be obtained from the holotype, topotypic specimens should be analyzed, an approach that we used for 19 of doi:10.1371/journal.pone.0038063.t002 the species examined in this study. However, the analysis of type specimens is clearly preferable because many species descriptions New Distribution and Life Stage Records lack detailed information on collection locality (e.g. state, province New distribution records were discovered for Caenis anceps or sometimes just ‘‘North America’’). When the type series has McDunnough, Maccaffertium smithae (Traver), Nixe joernensis (Bengts- been destroyed, the designation of a barcoded neotype will provide son), Acentrella nadineae McCafferty, Waltz & Webb, Acentrella a pathway to allow use of existing names and the description of lapponica Bengtsson, Isonychia rufa McDunnough and Baetis foemina new taxa [29–31]. For species with degraded types and ambiguous Ide (Tables S1, S3). New life stage associations include the larvae type locales, it may be useful to choose a specimen closely of Baetis persecutor McDunnough, Procloeon fragile (McDunnough) matching the morphological type concept as an informal ‘‘barcode and Isonychia berneri Kondratieff & Voshell; descriptions of these life type’’. stages are beyond the scope of this paper and will be treated While obtaining barcodes from type specimens will aid in the elsewhere. application of names to barcode clusters, further taxonomic work integrating multiple lines of evidence may be required to test Discussion current species hypotheses. For example, our results show that additional examination of the North American Baetidae and This paper presents the largest barcoding dataset of any order of Ephemerellidae is required as they both contain large numbers of aquatic insects, providing records for more than 4000 individuals species with multiple barcode clusters, high intraspecific morpho- from over 350 species. The analysis of both larvae and adults from logical variation, and many interim species identifications. DNA several localities and some topotypes enabled identification with barcoding results cannot only serve as a guide of where to focus less ambiguity than in the past. This allowed the correction of these efforts, but also be used as part of an iterative revisionary previously misidentified species and strengthened knowledge of the process [32] together with morphological, ecological, and behav- levels of COI variation both within and between species. Our ioral characteristics to achieve stable, robust species hypotheses. results confirm that levels of sequence divergence among closely Such refined species hypotheses will greatly improve our ability to allied species are generally high. For example, congeneric species determine and communicate the ecological characteristics of of North American Ephemeroptera showed a mean barcode a species, such as phenology and tolerance to pollutants, and in divergence of 13.9%, a much higher value than the 7–8% turn improve our ability to monitor ecological changes. This can divergence reported for congeneric species of Lepidoptera in both most effectively be achieved by involving taxonomists not only in North America [23] and Europe [24]. the identification of specimens for barcoding, but also in the design Our results indicate that much more taxonomic work is of barcoding projects and the selection and collection of specimens required on North American Ephemeroptera as many currently for further analysis [33]. recognized species include several highly divergent, often poly- This study has increased barcode coverage for North American phyletic, haplotypes, usually correlated with morphological Ephemeroptera from 121 to 354 species, or over 50% of the differentiation among lineages. Most of these species have complex known fauna. Because the present library includes records for histories of synonymy, reflecting a 60 year trend in North nearly all common species, most Ephemeroptera taken in routine American mayfly systematics towards inclusive species concepts. biomonitoring samples may now be rapidly and accurately This trend was driven by the observation of individuals identified through DNA barcoding, albeit with the proviso that morphologically intermediate between named species, or by further taxonomic work is needed to clarify species boundaries. observations suggesting that members of one species fell within Our progress in constructing a barcode library for North ‘the expected range of variation’ of another. Nearly all of these American Ephemeroptera further indicates the feasibility of decisions of synonymy were based only on limited morphological gaining global coverage both for this order and for other key studies, without consideration of biogeographic, ecological, groups of aquatic insects. behavioral or molecular data. While this fusion of species has simplified morphology-based identifications, it now seems likely Materials and Methods that this trend often ignored biological reality. For example, the four species in the lata complex were all synonymized Specimen Collection and Sequencing under Drunella lata (Morgan) based on morphological study [25], Detailed collection data are included in Table S3, and are also but morphometric, ecological, and allozyme data indicates at least available on BOLD (www.boldsystems.org) in the Virtual Project - three species in the northeastern United States [26]. Our results Ephemeroptera (mayflies) of North America - Phase I (DATA- confirm this conclusion and amplify it by indicating that D. SET-EPNA1). Specimens were identified following currently longicornis (Traver) likely also represents a valid species in the accepted taxonomy at Mayfly Central (http://www.entm. complex and that there is a fifth species in the southeastern United purdue.edu/mayfly/na-species-list.php), except the Arthropleidae States (Figure S1). was included in the Heptageniidae because of recent molecular Our results reveal that many species currently treated as and integrated phylogenies [34,35]. When discrepancies were de- synonyms may well represent valid species. A critical step in the tected between barcode results and morphological identifications,

PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e38063 DNA Barcoding North American Ephemeroptera specimens were reexamined. When specimens could not be Kimura-2-Parameter (K2P) distances were calculated in MEGA5 identified morphologically (e.g. certain females, some larvae), they using the pairwise deletion option and a Neighbor Joining tree was were assigned the same name as expertly identified specimens with generated. either the same or a closely similar (,2% divergence) COI haplotype. Specimen(s) which formed a unique barcode cluster, Supporting Information but which could not be identified morphologically, were assigned a provisional name structured in a consistent fashion (generic Figure S1 Neighbour Joining tree using Kimura-2-Parameter name followed by a species name composed of ‘sp.’ followed by distance for COI DNA sequences from 4065 individuals of North initials of the taxonomist e.g. Heptagenia sp.LJ1; exceptions include American Ephemeroptera. Specimens and species with topotypes previously published provisional names e.g. Acerpenna sp. CHU1 are indicated with ‘*’; specimens and species from the same and those from California identified as part of an ongoing general area as the type locality are indicated with ‘#’. Southern California Coastal Water Research Project e.g. Drunella (PDF) sp.CA1). Table S1 Species-level summary of K2P distance, sample Topotypes, specimens collected from the type locality and distribution, and sample size for North American Ephemeroptera. which have a high certainty of representing the type concept of All distributional records use standard 2-letter (or 3-letter, for a species, were sequenced for the following species: Acentrella Mexico) postal abbreviations. MNID = minimum interspecific parvula Ameletus amador A. andersoni A. bellulus A. , Mayo, Zloty, Zloty, K2P distance, MXID = maximum intraspecific K2P distance, pritchardi Zloty, Baetis adonis Traver, Caenis eglinensis Pescador & species with maximum intraspecific sequence divergence $5.0% Richard, Cercobrachys cree Sun, Webb & McCafferty, Drunella grandis are indicated with ‘*’. (Eaton), Epeorus albertae (McDunnough), Ephemerella dorothea infre- (DOC) quens, Eurylophella doris (Traver), E. oviruptis Funk, E. poconoensis Funk and Paraleptophlebia kirchneri Kondratieff & Durfee. Individuals from Table S2 Updated identifications for Ephemeroptera specimens near the type locality were sequenced for Baetodes tritus Cohen & with published barcode records. Allen, Camelobaetidius trivialis Allen & Chao [currently considered (DOC) a synonym of C. warreni (Traver & Edmunds)], Heptagenia julia Table S3 Collection data for all barcoded specimens of Traver, and Susperatus prudens (McDunnough). Ephemeroptera. Each barcode sequence (.400 bp) for North American (XLS) Ephemeroptera from previous publications (150 - [2], 13 - [19], 1 -[1], 414 - [12], 564 - [20]) were assembled and their source Text S1 Taxonomic notes on selected North American Ephe- specimen was reexamined, whenever possible, to confirm its meroptera species with barcode records. identification. In addition, barcodes were generated for another (DOC) 3024 specimens at the Canadian Centre for DNA Barcoding using standard protocols for DNA extraction, polymerase chain reaction Acknowledgments (PCR) and sequencing [36,37]. PCR was performed using the standard LCO1490/HCO2198 and/or LepF1/LepR1 primers Special thanks are given to Parks Canada for help with access to field sites with M13 tails. When these protocols failed to recover a sequence, in Cape Breton Highlands National Park, and to the Stroud Water Research Center, Pennsylvania, the Southern California Coastal Water the primer set LCO1490 and MEPTR1-t1 was used to recover Research Project, Costa Mesa, California, and the Aquatic Bioassessment a 325 bp segment from the 59 end of the barcode region [20]. Lab, California State University, Chico for providing California specimens Sequences were obtained from the following Barcode of Life and valuable discussion. Additional specimens were provided by the Data systems (BOLD systems: http://www.boldsystems.org) pro- Highlands Biological Station, North Carolina. S. Bateson, Biodiversity ject codes: ELPYO, BBEPT, CPMAY, ECEPH, FAMAY, Institute of Ontario, provided assistance in production of trees and maps. SMMAY, INHSE, LJMAY, LJGSM, BKMAY, ABMAY, MBMAY, NYMAY, ONMAY, SKMAY, USMAY, PUMAY, Author Contributions CFWIA, CFWIB, CFWIC, CFWID, CFWIE, CFWIF, CFWIG, Conceived and designed the experiments: JMW LMJ DHF XZ BK CJG CFWIH, CFWII, SWRCE, SWRCD, PRESV, GSEPT, SWAMI, RED DJB BR IP PDNH. Performed the experiments: JMW LMJ DHF WEAI, SBEP, NBMAY, HIEPT, HIMXD. XZ BK CJG RED DJB BR IP. Analyzed the data: JMW LMJ DHF XZ COI sequences were aligned in MEGA 5 [38] using the BK CJG RED DJB BR IP. Contributed reagents/materials/analysis tools: integrated ClustalX method with default parameters. All se- JMW LMJ DHF XZ BK CJG RED DJB BR IP PDNH. Wrote the paper: quences were examined for the presence of stop codons and indels. JMW PDNH LMJ DHF.

References 1. Hebert PDN, Cywinska A, Ball SL, DeWaard JR (2003) Biological identifica- of the North American Benthological Society 26: 719–742. doi:10.1899/06- tions through DNA barcodes. Proceedings of the Royal Society of London Series 089.1. B-Biological Sciences 270: 313–321. doi:10.1098/rspb.2002.2218. 6. Rosenberg D, Resh VH (1993) An introduction to freshwater biomonitoring and 2. Ball SL, Hebert PDN, Burian SK, Webb JM (2005) Biological identifications of benthic invertebrates. In: Rosenberg DM, Resh VH, eds. Freshwater mayflies (Ephemeroptera) using DNA barcodes. Journal of the North American Biomonitoring and Benthic Macroinvertebrates. New York: Chapman and Benthological Society 24: 508–524. Hall. pp 1–9. 3. Zhou X, Robinson JL, Geraci CJ, Parker R, Flint OS, et al. (2011) Accelerated 7. Pfrender ME, Hawkins CP, Bagley M, Courtney GW, Creutzburg BR, et al. construction of a regional DNA-barcode reference library: caddisflies (Trichop- (2010) Assessing macroinvertebrate biodiversity in freshwater ecosystems: tera) in the Great Smoky Mountains National Park. Journal of the North advances and challenges in DNA-based approaches. The Quarterly Review of American Benthological Society 30: 131–162. doi:10.1899/10-010.1. Biology 85: 319–340. 4. Mynott JH, Webb JM, Suter PJ (2011) Adult and larval associations of the alpine 8. Baird DJ, Sweeney BW (2011) Applying DNA barcoding in benthology: the state stonefly genus Riekoperla McLellan (Plecoptera: Gripopterygidae) using mito- of the science. Journal of the North American Benthological Society 30: chondrial DNA. Invertebrate Systematics 25: 11–21. 122–124. doi:10.1899/10-154.1. 5. Zhou X, Kjer KM, Morse JC (2007) Associating larvae and adults of Chinese 9. Baird DJ, Pascoe TJ, Zhou X, Hajibabaei M (2011) Building freshwater Hydropsychidae caddisflies (Insecta:Trichoptera) using DNA sequences. Journal macroinvertebrate DNA-barcode libraries from reference collection material:

PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e38063 DNA Barcoding North American Ephemeroptera

formalin preservation vs specimen age. Journal of the North American 24. Hausmann A, Haszprunar G, Segerer AH, Speidel W, Behounek G, et al. (2011) Benthological Society 30: 125–130. doi:10.1899/10-013.1. Now DNA-barcoded: the butterflies and larger moths of Germany. Spixiana 34: 10. Hajibabaei M, Shokralla S, Zhou X, Singer GAC, Baird DJ (2011) 47–58. Environmental barcoding: a next-generation sequencing approach for biomo- 25. Jacobus LM, McCafferty WP (2004) Revisionary contributions to the nitoring applications using river benthos. PLoS ONE 6: e17497. doi:10.1371/ genus Drunella (Ephemeroptera: Ephemerellidae). Journal of the New York journal.pone.0017497. Entomological Society 112: 127–147. doi:10.1664/0028- 11. Pilgrim EM, Jackson S, Swenson S, Turcsanyi I, Friedman E, et al. (2011) 7199(2004)112[0127:RCTTGD]2.0.CO; 2. Incorporation of DNA barcoding into a large-scale biomonitoring program: 26. Funk DH, Sweeney BW, Jackson JK (2008) A taxonomic reassessment of the opportunities and pitfalls. Journal of the North American Benthological Society Drunella lata (Morgan) species complex (Ephemeroptera:Ephemerellidae) in 30: 217–231. doi:10.1899/10-012.1. northeastern North America. Journal of the North American Benthological 12. Sweeney BW, Battle JM, Jackson JK, Dapkey T (2011) Can DNA barcodes of Society 27: 647–663. doi:10.1899/08-026.1. stream macroinvertebrates improve descriptions of community structure and 27. Hajibabaei M, Smith MA, Janzen DH, Rodriguez JJ, Whitfield JB, et al. (2006) water quality? Journal of the North American Benthological Society 30: A minimalist barcode can identify a specimen whose DNA is degraded. 195–216. doi:10.1899/10-016.1. Molecular Ecology Notes 6: 959–964. doi:10.1111/j.1471-8286.2006.01470.x. 28. Shokralla S, Zhou X, Janzen DH, Hallwachs W, Landry J-F, et al. (2011) 13. Cao Y, Hawkins CP (2011) The comparability of bioassessments: a review of Pyrosequencing for mini-barcoding of fresh and old museum specimens. PLoS conceptual and methodological issues 1. Journal of the North American ONE 6: e21252. doi:10.1371/journal.pone.0021252. Benthological Society 30: 680–701. doi:10.1899/10-067.1. 29. James SW, Porco D, Decae¨ns T, Richard B, Rougerie R, et al. (2010) DNA 14. Lenat DR, Resh VH (2001) Taxonomy and stream ecology: the benefits of barcoding reveals cryptic diversity in Lumbricus terrestris L., 1758 (Clitellata): genus- and species-level identifications. Journal of the North American resurrection of L. herculeus (Savigny, 1826). PloS one 5: e15629. doi:10.1371/ Benthological Society 20: 287. doi:10.2307/1468323. journal.pone.0015629. 15. Feio MJ, Reynoldson TB, Grac¸a MAS (2006) The influence of taxonomic level 30. Favret C, Miller GL (2011) The neotype of the cotton aphid (Hemiptera: on the performance of a predictive model for water quality assessment. Water Aphididae: Aphis gossypii Glover 1877). Proceedings of the Entomological Society Research 376: 367–376. doi:10.1139/F05–221. of Washington 113: 119–126. 16. Poff NL, Olden JD, Vieira NKM, Finn DS, Simmons MP, et al. (2006) 31. Hausmann A, Hebert PDN, Mitchell A, Rougerie R, Sommerer M, et al. (2009) Functional trait niches of North American lotic insects: traits-based ecological Revision of the Australian Oenochroma vinaria Guene´e, 1858 species-complex applications in light of phylogenetic relationships. Journal of the North (Lepidoptera: Geometridae, Oenochrominae): DNA barcoding reveals cryptic American Benthological Society 25: 730–755. diversity and assesses status of type specimen without dissection. Zootaxa 2239: 17. Lenat DR (1993) A biotic index for the southeastern United States: derivation 1–21. and list of tolerance values, with criteria for assigning water-quality ratings. 32. Yeates DK, Seago A, Nelson L, Cameron SL, Joseph L, et al. (2011) Integrative Journal of the North American Benthological Society 12: 279–290. taxonomy, or iterative taxonomy? Systematic Entomology 36: 209–217. 18. Lenat DR, Penrose DL (1996) History of the EPT taxa richness metric. Bulletin doi:10.1111/j.1365-3113.2010.00558.x. of the North American Benthological Society 13: 305–306. 33. DeWalt RE (2011) DNA barcoding: a taxonomic point of view. Journal of the 19. Webb JM, Sun L, McCafferty WP, Ferris VR (2007) A new species and new North American Benthological Society 30: 174–181. doi:10.1899/10-021.1. synonym in Heptagenia Walsh (Ephemeroptera: Heptageniidae: Heptageniinae) 34. Ogden TH, Whiting MF (2005) Phylogeny of Ephemeroptera (mayflies) based based on molecular and morphological evidence. Journal of Science 7: 63. on molecular evidence. Molecular Phylogenetics and Evolution 37: 625–643. doi:10.1673/031.007.6301. doi:10.1016/j.ympev.2005.08.008. 20. Zhou X, Adamowicz SJ, Jacobus LM, DeWalt RE, Hebert PDN (2009) 35. Ogden TH, Gattolliat JL, Sartori M, Staniczek AH, Solda´n T, et al. (2009) Towards a comprehensive barcode library for arctic life - Ephemeroptera, Towards a new paradigm in mayfly phylogeny (Ephemeroptera): combined Plecoptera, and Trichoptera of Churchill, Manitoba, Canada. Frontiers in analysis of morphological and molecular data. Systematic Entomology 34: Zoology 6: 30. doi:10.1186/1742-9994-6-30. 616–634. doi:10.1111/j.1365-3113.2009.00488.x. 21. Zhou X, Jacobus LM, DeWalt RE, Adamowicz SJ, Hebert PDN (2010) 36. Ivanova NV, DeWaard JR, Hebert PDN (2006) An inexpensive, automation- Ephemeroptera, Plecoptera, and Trichoptera fauna of Churchill (Manitoba, friendly protocol for recovering high-quality DNA. Molecular Ecology Notes 6: Canada): insights into biodiversity patterns from DNA barcoding. Journal of the 998–1002. doi:10.1111/j.1471-8286.2006.01428.x. North American Benthological Society 29: 814–837. doi:10.1899/09-121.1. 37. DeWaard JR, Ivanova NV, Hajibabaei M, Hebert PDN (2008) Assembling 22. Alexander LC, Delion M, Hawthorne DJ, Lamp WO, Funk DH (2009) DNA barcodes: analytical protocols. In: Martin CC, ed. Methods in Molecular Biology. Volume 410: Environmental Genomics. Totowa, New Jersey: Humana Mitochondrial lineages and DNA barcoding of closely related species in the Press. pp 275–283. mayfly genus Ephemerella (Ephemeroptera: Ephemerellidae). Journal of the North 38. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, et al. (2011) MEGA5: American Benthological Society 28: 584–595. doi:10.1899/08-150.1. molecular evolutionary genetics analysis using maximum likelihood, evolution- 23. Hebert PDN, DeWaard JR, Landry J-F (2010) DNA barcodes for 1/1000 of the ary distance, and maximum parsimony methods. Molecular Biology and kingdom. Biology letters 6: 359–362. doi:10.1098/rsbl.2009.0848. Evolution 28: 2731–2739. doi:10.1093/molbev/msr121.

PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e38063